1. Field of the Invention
The present invention relates to improved methods for targeting boron atoms to tumor cells for effecting boron neutron capture therapy (BNCT). BNCT is a binary system designed to deliver ionizing radiation to tumor cells by neutron irradiation of tumor-localized boron-10 atoms. In the present invention, the cancer cells are pre-targeted, for example, with a multivalent antibody conjugate wherein at least one antibody or antibody fragment specifically targets tumor cells and at least one antibody or antibody fragment specifically binds to a boron compound. Then, the boron compound is administered and is bound by the multivalent antibody conjugate localized at the cancer site. The localized boron may then be irradiated, thereby effecting treatment of the tumor cells.
2. Description of Related Art
BORON NEUTRON CAPTURE THERAPY
Boron neutron capture therapy (BNCT) is based on the nuclear reaction which occurs when a stable isotope, B-10 (present in 19.8% natural abundance), is irradiated with thermal neutrons to produce an alpha particle and a Li-7 nucleus. These particles have a path length of about one cell diameter, resulting in high linear energy transfer. Just a few of the short-range 1.7 MeV alpha particles produced in this nuclear reaction are sufficient to target the cell nucleus and destroy it. Barth et al., Cancer, 70: 2995-3007 (1992). Since the .sup.10 B(n,.alpha.).sup.7 Li reaction will occur, and thereby produce significant biological effect, only when there is a sufficient fluence (number) of thermal neutrons and a critical amount of B-10 localized around or within the malignant cell, the radiation produced is localized. The neutron capture cross section of B-10 far exceeds that of nitrogen and hydrogen found in tissues, which also can undergo capture reactions, (relative numbers: 1 for N-14, 5.3 for H-1, and 11560 for B-10), so that once a high concentration differential of B-10 is achieved between normal and malignant cells, only the latter will be affected upon neutron irradiation. This is the scientific basis for boron neutron capture therapy. Barth et al., supra; Barth et al. Cancer Res., 50: 1061-70 (1990); Perks et al., Brit. J. Radiol., 61: 1115-26 (1988).
Nuclear reactors are the source of neutrons for BNCT. Thermal neutron beams with energies in the range of 0.023 eV, used in early experiments for treating brain tumors, are easily attenuated by tissues, and are poorly penetrating. More recent advances with neutrons of intermediate energy (epithermal neutrons, 1-10,000 eV energy) have led to the consensus for its use in planned clinical trials in the US and Europe. Alam et al., J. Med. Chem., 32: 2326-30 (1989). Fast neutrons with a probable energy of 0.75 MeV are of little use in BNCT.
Original calculations estimated that a boron concentration of 35-50 .mu.g per gram of tumor, or 10.sup.9 B-10 atoms per tumor cell, would be necessary to sustain a cell-killing nuclear reaction with thermal neutron fluences of 10.sup.12 -10.sup.13 n.cm.sup.-2. Fairchild et al., Int. J. Radiat. Oncol. Biol. Phys., 11: 831 (1985). These calculations were based on uniformly distributed boron, as seen with non-specific boronated compounds. For antibody-based boron agents, assuming saturation of all surface antigens on the tumor cell, this level of boron requirement translates to about 1000 atoms per antibody molecule. However, more recent Monte Carlo calculations led to the analysis that for a non-internalizing antibody, boron loading could be as low as 300 atoms per MAb molecule. Kalend et al, Med. Phys., 18: 662 (1991); Zamenhof et al., J. Nat'l Cancer Inst., 84: 1290-91 (1992).
This was based on the following rationale: for tumor cells exhibiting a nucleus-to-cell volume ratio of 0.5 and an effective cell diameter of 10 .mu.m, three B-10 fissions on the cell surface would produce at least one heavy particle trajectory into the nucleus. Assuming saturation of antigen sites on the cell surface, it was deduced that under these conditions just 300 atoms per antibody molecule would suffice to bring about the three fission reactions on the tumor cell surface. The present invention describes a method which can attach a 20-fold greater number of boron atoms per MAb than these prior methods entailed.
Historically, BNCT was first employed for the treatment of glioblastoma (a fatal form of brain tumor) and other brain tumors at a time when tumor specific substances were almost unknown. Hatanaka et al., in BORON NEUTRON CAPTURE THERAPY FOR TUMORS, pp.349-78 (Nishimura Co., 1986). One of the first boronated compounds employed, a sulfhydryl-containing boron substance called sodium borocaptate or BSH (Na.sub.2, B.sub.12 H.sub.11 SH), crosses the blood-brain barrier to localize in brain, and this has been the anatomical basis for neutron capture therapy of brain tumors. Clinical trials have been carried out, or are scheduled, for the treatment of gliomas in Japan, the US and Europe. Barth et al., Cancer, supra. Problems with previous inorganic boron therapy methods was that the boron reached both targeted and non-target areas. Accordingly, when the boron was irradiated, healthy cells as well as cancerous cells were destroyed.
The BNCT concept has been extended to other cancers, spurred on by the discovery of a number of tumor-localizing substances, including tumor-targeting monoclonal antibodies. For instance, boronated amino acids such as p-boronophenylalanine accumulated in melanoma cells. The potential of using boronated monoclonal antibodies directed against cell surface antigens, such as CEA, for BNCT of cancers has been demonstrated. Ichihashi et al., J. Invest. Dermatol, 78: 215-18 (1982); Goldenberg et al., P.N.A.S., USA, 81:560-63 (1984); Mizusawa et al. P.N.A.S., USA, 79: 3011-14 (1982); Barth et al., Hybridoma, 5(supp. 1): 543-5540 (1986); Ranadive et al. Nuci. Med. Biol., 20: 663-68 (1993). However, heavily boronated antibodies failed to target tumor in vivo in animal models. Alam et al., supra; Barth et al., Bioconjugate Chem., 5: 58-66 (1994).
Success with BNCT of cancer requires methods for localizing a high concentration of boron-10 at tumor sites, while leaving non-target organs essentially boron-free. Non-antibody boronated compounds which accumulate in tumor preferentially, but not specifically, have the disadvantage that tumor-to-blood and tumor-to-organ ratios are often less than ideal, with the result that damage to normal organs could occur during irradiation with neutron beams.
In the case of antibodies, the perceived need to load the same with 1000 boron atoms per antibody molecule has led to the design of a variety of heavily boronated antibodies using, for instance, polylysine, dendrimer or dextran as intermediate carriers of boron clusters. Alam et al., supra; Barth et al., Bioconjugate Chem., supra. Although in many instances some antigen-binding was found to be retained in vitro, these boronated conjugates predominantly localized in liver with little accretion in tumor in in vivo animal tumor models.
Thus, there is need for a method of targeting boron atoms to tumor cells that is able to deliver a large amount of boron atoms to tumor sites, while leaving noncancerous sites relatively boron-free.
PRE-TARGETING
The concept of pre-targeting for in vivo imaging application was proposed by Hnatowich et al., J. Nucl. Med., 28: 1294-1302 (1987), and was later examined from a theoretical viewpoint. Van Osdol et al., J. Nucl. Med., 34: 1552-64 (1993). Pre-targeting has been recently reported to have resulted in very encouraging preclinical results with yttrium-90 radioimmmunotherapy. Axworthy et al., J. Immunother., 16: 158 (1994). U.S. application Ser. No. 07/933,982 (filed Aug. 21, 1992, issue fee paid Dec. 28, 1995), U.S. Pat. No. 5,482,698, U.S. application Ser. No. 08/409,960 (filed Mar. 25, 1995, pending), and U.S. application Ser. No. 08/486,166 (filed Jun. 7, 1995, pending) also disclose various pre-targeting methods. The contents of all of these references are incorporated herein in their entirety by reference.
Therapy requires a high absolute accretion of the therapeutic agent at the cancer site, as well as a reasonably long duration of uptake and binding. High background levels of non-targeting antibody have long been recognized as a major impediment to high target: background ratios being achieved. To overcome this impediment, various methods have been developed, such as those described in Hansen et al., U.S. Pat. No. 3,927,193 and Goldenberg, U.S. Pat. Nos. 4,331,647, 4,348,376, 4,361,544, 4,468,457, 4,444,744, 4,460,459, 4,460,561, 4,624,846 and 4,818,709, the disclosures of all of which are incorporated herein by reference.
Pre-targeting methods using biotin/avidin approaches are described, for example, in Hnatowich et al., J. Nucl. Med. 28: 1294, 1987; Oehr et al., J. Nucl. Med. 29: 728, 1988; Klibanov et al., J. Nucl. Med. 29: 1951, 1988; Sinitsyn et al., J. Nucl. Med. 30: 66, 1989; Kalofonos et al, J. Nuc. Med. 31: 1791, 1990; Schechter et al., Int. J. Cancer 48: 167, 1991; Paganelli et al., Cancer Res. 51: 5960, 1991; Paganelli et al., Nucl. Med. Commun. 12: 211, 1991; Stickney et al., Cancer Res. 51: 6650, 1991; and Yuan et al., Cancer Res. 51: 3119, 1991; all incorporated herein in their entirety by reference.
Pre-targeting a target site with a targeting protein, such as an antibody or an antibody fragment, conjugated to an antibody or antibody fragment specific for the second conjugate also has been described. U.S. Pat. Nos. 5,274,076, 4,863,713 and 5,256,395.
These methods involve pre-targeting a target site, such as a tumor or lesion, with a targeting protein, such as an antibody or antibody fragment, conjugated to one member of a binding pair, such as biotin, avidin, or a second antibody, whereby the antibody conjugate localizes at the target site. Then, a conjugate of a detection or therapeutic agent, such as a radioisotope, and the complementary member of the binding pair, such as avidin, biotin, or a corresponding hapten is administered. The binding affinity between the members of the binding pair causes the second conjugate to localize at the target site, where the first conjugate already is bound.
In some of these methods, an intermediate clearing or localizing step is used. In this case, the first conjugate comprises one member of the binding pair (for example, biotin), the clearing and localizing agent may comprise the other member of the binding pair (for example, avidin), and the second conjugate comprises the same member of the binding pair as the first (for example, biotin). Other clearing agents, such as antibodies, also have been described.
There is a need for a method of targeting boron atoms to tumor cells that obtains high tumor:non-tumor ratios of the boron atoms, and that delivers sufficient amounts of boron atoms to tumor sites in an efficient manner. Compositions suitable for use in such a method also are needed.